Several studies describe gastrointestinal tract (GIT) melatonin (MEL) synthesis from 5-HT, which itself derives from the essential amino acid L-tryptophan (L-trp) in the intestine. Supplementing L-trp and MEL through diet has shown social-response effects and suppresses neuroendocrine stress in teleosts. In this study, the effects of a MEL and L-trp-supplemented diet on the endocrine intestinal function and enzymatic response activity of two salmonid species were examined. To assess the possible effect of L-trp and MEL on intestinal serotonin content and digestive enzyme activity, three L-trp-supplemented diets and three MEL-supplemented diets were orally administered to a group of Salmo salar and Oncorhynchus kisutch for seven days under normal density conditions. Plasma biochemistry (cortisol, L-trp, MEL) as well as enzyme activity (amylase, lipase, and total protease) and serotonin content were measured in the pyloric caeca, midgut, and hindgut. Plasma L-trp levels were found to be directly related to L-trp supplemented diet levels. Similarly, MEL supplementation increased plasma MEL levels, and the presence of MEL in both salmon species resulted in a significant interaction with cortisol concentrations in plasma, and the highest concentrations of L-trp caused an increased GIT content for 5-HT in S. salar. No differences were seen in the GIT content for 5-HT for the L-trp supplemented diets in O. kisutch. An inhibitory effect was found on digestive enzymes in the supplemented diets of both salmonid species. In general, the presence of MEL in the diet reduced cortisol levels; diets supplemented with L-trp and MEL had either a stimulatory or inhibitory effect on digestive enzyme activity, which seemed to be indirect and tissue dependent.